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Fig. 1 | BMC Biotechnology

Fig. 1

From: New tools to convert bacterial artificial chromosomes to a self-excising design and their application to a herpes simplex virus type 1 infectious clone

Fig. 1

Construction and analysis of pHSVF-BFP. a Flow diagram of two-step recombination (En Passant) used to insert a TagBFP expression cassette into the vector backbone of pYEbac102 in E. coli, and subsequent Cre-based removal of the BAC vector from the viral genome in mammalian cells. The expression cassette was PCR amplified from the pEP-TagBFP-in template and recombined into the BAC vector backbone by lambda RED recombination using kanamycin resistance as selective pressure. In a second recombination step, the kanamycin resistance gene (aphA1) was removed based on partially duplicated sequences in the flanking TagBFP coding sequence (red boxes), which simultaneously established the contiguous BFP coding sequence and resulted in pHSVF-BFP. Transfection of pHSVF-BFP into Vero cells produced the vHSVF-BFP virus that stably expressed blue fluorescence, which was further expanded by a second passage (P2) on Vero cells. Transfection and serial passage in Vero-cre cells produced HSV-1 lacking the BAC backbone and associated fluorescence but retaining a single loxP site (black circles) between the UL3 and UL4 genes. b Excision of the pBeloBAC vector from vHSVF-BFP was monitored by fluorescent plaque assay. Following transfection of Vero cells (P1), vHSVF-BFP was successively passaged on Vero-cre cells for a second (P2cre), third (P3cre), and fourth round (P4cre), or on Vero cells that did not express Cre recombinase for a second round (P2). Plaques produced from each harvest were scored as positive (blue) or negative (black) for fluorescence. The data are a composite of three independent experiments consisting of >40 plaques scored per experiment. Error bars are SD

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